Question

The vector, shown in the figure, has promoter and RBS sequences in the 300 bp region between the restriction sites for enzymes X and Y. There are no other sites for X and Y in the vector. The promoter is directed towards the Y site. The insert containing only an ORF provides 3 fragments after digestion with both enzymes X and Y. The ORF is cloned in the correct orientation in the vector using the single restriction enzyme Y. The size of the largest fragment of the recombinant plasmid expressing the ORF upon digestion with enzyme X is ........... bp. (answer in integer) 

Hint:

When cloning a gene, the size of the largest fragment after digestion with restriction enzymes is the sum of the vector region and the insert size, assuming the insert is in the correct orientation.

Answer 1

Step 1: Understanding the figure.
The vector contains a 300 bp region between the restriction sites for enzymes X and Y. The promoter and RBS sequences are within this 300 bp region, and there are no other restriction sites for X and Y.
The insert with the ORF is placed in the vector in the correct orientation, and upon digestion with enzymes X and Y, 3 fragments are generated.
Step 2: Analyzing the digestion.
Since enzyme Y is used to cut the vector and the insert is in the correct orientation, the fragments generated after digestion with X are as follows:
- The fragment created by enzyme X is the largest one, which includes the 300 bp region of the vector plus the ORF.
- The other two fragments are smaller, and one of them corresponds to the promoter region and the RBS.
The largest fragment will be the one containing the 300 bp region plus the insert, which has a size of 1.2 kb (as given in the figure).
Final Answer: \[ \boxed{1200 \, \text{bp}} \]